Fibrous collagens are integral parts of the extracellular matrix that support tissue integrity and maintain the cellular microenvironment for normal physiological functions. Collagens I-III, the major isoforms of the fibrous collagen protein family, are synthesized as procollagen precursors containing N-terminal and C-terminal propeptides. The procollagens are post-translationally modified by proline hydroxylation, and secreted into the peri-vascular space for further processing. N-terminal propeptides of the collagens are subsequently cleaved by proteinases of the ADAMTS (A Distintegrin And Metalloproteinase with ThromboSpondin repeats) family, while the C-terminal propeptides are processed by the Tolloid family of metalloproteases, which include BMP1, TLL1 and TLL2 (Hopkins, D. R. et al., Matrix Biology, 2007, 26, 508-523). The cleavage of both N-terminal and C-terminal propeptides allows further maturation of the collagen, leading to cross-linking at lysine residues and formation of insoluble fibrillar structures (Shoulders, M. D. et al., Annual Review of Biochemistry, 2009, 78, 929-958).
Whereas the BMP1, TLL1 and TLL2 proteins are encoded by separate genes, this family also includes isoforms of BMP1, including multiple isoforms of BMP1 that result from alternative splicing of the same gene product (see e.g., Takahara, K., et al., The Journal of Biological Chemistry, 1994, 269. 32572-32578; and Cvetjeticanin, B. et al., Medical Hypotheses, 2014, 83, 656-658). The originally discovered form of BMP1 is designated BMP-1-1 or BMP1-1. Other BMP1 isoforms encoded by splice variant RNA transcripts have been described at the transcriptional level and designated with sequential suffixes, e.g., as BMP-1-2, BMP-1-3, BMP-1-4, BMP-1-5, BMP-1-6, and BMP-1-7 (see, e.g., Wozney et al., Science (1988), 242: 1528-1534; Kessler et al., Science, (1996) 271: 360-362; Li et al., Proc. Natl. Acad. Sci. USA (1996), 93: 5127-5130; Janitz et al., J. Mol. Med. (1998), 76: 141-146; Takahara et al., J. Biol. Chem. (1994), 269: 32572-32578; and Ge and Greenspan, Birth Defect Res. (2006), 78: 47-68).
A number of BMP1 isoforms have also been confirmed at the protein level as circulating in the blood of patients with various diseases and in healthy humans (see, e.g., International Patent publication Nos. WO2008/011193 A2 and WO2013/163479 A1, and Grgurevic et al., J. Am. Soc. Nephrol. (2011), 21:681-692). In addition, the role of BMP1 in processing procollagen leading to fibrosis and scar tissue in a variety of diseases as well as the discovery of blood profiles comprising individual BMP1 isoforms in patients with various diseases has made BMP1 an attractive target for developing new therapies (see, e.g. WO2008/011193 A2; WO2013/163479 A1; Grgurevic et al., J. Am. Soc. Nephrol. (2011), 21:681-692, Cvetjeticanin, B. et al., Medical Hypotheses, 2014, 83, 656-658; and Turtle et al., Expert Opin. Ther. Patents (2004), 14(8):1185-1197).
Excessive production of extracellular matrix (ECM) proteins, including collagen, can lead to fibrotic pathologies in various organs or tissues that may be associated with increased tissue rigidity, parenchymal replacement, aberrant electrical conductance, sclerotic wound healing (e.g. infarction and burns), and/or abnormal cell-cell interactions. For example, increased fibrosis and collagen production are consistently observed in patients with acute and chronic cardiac diseases, e.g., heart failure, arrhythmias, hypertrophic cardiomyopathy, and myocardial infarction (Lopez, B. et al., Circulation, 2010, 121, 1645-1654; Ho, C. Y., et al., New England Journal of Medicine, 2010, 363, 552-563; Kostin, S. et al., Cardiovascular Research, 2002, 54, 361-379; See, F., et al., Current Pharmaceutical Design, 2005, 11, 477-487; Cvetjeticanin, B. et al. Medical Hypotheses, 2014, 83, 656-658), chronic obstructive pulmonary disease (“COPD”) (Salazar, L. M., et al., Lung, 2011, 189, 101-109), liver cirrhosis and nonalcoholic steatohepatitis (“NASH”) (Bataller, R., et al., Journal of Clinical Investigation, 2005, 115, 209-218), idiopathic pulmonary fibrosis (Chakraborty, S, et al., Expert Opin Investig Drugs, 2014, 23, 893-910), collagen vascular diseases, e.g. systemic lupus erythematosus, rheumatoid arhthritis and scleroderma (Eckes, B., et al., J Mol Med, 2014, 92, 913-924), muscular dystrophies (e.g., Serrano, A. C., et al., Experimental Cell Research, 2010, 316, 3050-3058; Klingler, W., et al., Acta Myoligica, XXXI, 2012, 184-195), chronic kidney disease (Liu, Y., Nature Reviews Nephrology, 2011, 7, 684-696), acute kidney injury (Molitoris, B., The Journal of clinical Investigation, 2014, 124, 2355-2363; Venkatachalam, M. A. et al., Am J Physiol Renal Physiol 298: F1078-F1094, 2010), diabetic nephropathy (Sun, Y. M., et a., Biochemical and Biophysical Research Communications, 2013, 433, 359-361), keloids, wound healing, adhesions, hypertrophic and other scarring associated with, e.g. burns, surgery and other trauma (Meier K., et al., Expert Opinion on Emerging Drugs, 2006, 11, 39-47; Malecaze, F., et al., Investigative Opthalmology and Visual Science, 2014, 55, 6712-6721; van der Weer, W. et al., Burns, 2009, 35, 15-29), stroke, multiple sclerosis and spinal cord injury (Fernandez-Klett, F. and Piller, J. Brain Pathology, 2014, 24, 404-13; Rimar, D. et al., Arthritis & Rheumatology, Vol. 66, No. 3, March 2014, 726-730). Therefore, reducing excessive collagen production and maturation by targeting the BMP1, TLL1 and/or TLL2 pathway(s) can be an effective therapeutic strategy for treating fibrotic pathologies such as these diseases. This is supported by recent published studies using pharmacological agents that inhibit BMP1, TLL1 and/or TLL2 activity in cardiac and kidney disease models in small animals (Grgurevic, L., et al., Journal of the AmericanSociety of Nephrology, 2011, 21, 681-692; He, W., et al., Proceedings of the National Academy of Sciences, 2010, 107, 21110-21115; Cvetjeticanin, B. et al., Medical Hypotheses, 2014, 83, 656-658; International Patent publication Nos. WO2008/011193 A2 and WO2013/163479 A1).
The Tolloid family of metalloproteases (BMP1, TLL1 and TLL2) has additional substrates beyond collagens that may also contribute to its role in promoting ECM protein production. For example, the pro-form of lysyl oxidase 1 (LOX1) has been shown to be a substrate of BMP1, and cleavage by BMP1 enhances the LOX enzyme activity and thereby induces collagen cross-linking (Uzel, M. I., et al., Journal of Biological Chemistry, 2001, 276, 22537-22543). Thus, BMP1 also has a role in the development of pathological tissue stiffness via this mechanism, for example in glaucoma (Tovar-Vidales, T., et al., Investigative Ophthalmology & Visual Science, 2013, 54, 4741-4748) and in diastolic dysfunction in the heart (López, B., et al., American Journal of Physiology—Heart and Circulatory Physiology, 2010, 299, H1-H9). TGF-beta binding protein (LTBP) has also been shown to be cleaved by BMP1, allowing enhanced TGF-beta action to induce further collagen production (Ge, G., et al., Journal of Cell Biology, 2006, 175, 111-120). Regulation of TGF-beta by BMP1 may also play roles in other pathologies, such as control of cancer cell metastasis and invasion (Wu, X., et al. Oncogene, 2014, 33, 1506-1514). Similarly, BMP1, TLL1 and/or TLL2 also activate a broader range of other TGF-beta like molecules, such as BMPs 2 and 4, by proteolytically processing interacting proteins (Hopkins, D. R. et al., Matrix Biology, 2007, 26, 508-523). The combined actions of BMP1 and its various substrates suggest that BMP1, TLL1 and TLL2 are key regulators of tissue ECM production/maturation and that the members of the tolloid family of metalloproteases are particularly effective targets for anti-fibrosis therapeutic intervention.
BMP1, TLL1 and TLL2 may also affect other biological pathways via additional substrate processing. In particular, they may affect muscle biology via promoting activation of myostatin. Myostatin is a hormone that negatively regulates muscle growth (Lee, S. J., 2004, Annual Review of Cell & Developmental Biology, 20, 61-86). BMP1 has been demonstrated to cleave an inhibitory pro-peptide of myostatin and thus enhance myostatin activity (Wolfman N. M., et al., Proceedings of the National Academy of Sciences, 2003, 100, 15842-15846). Knockout of TLL2 in mice demonstrated enhanced muscle mass, thereby providing support for the connection between tolloid metalloprotease and myostatin (Lee, S. J., PLoS one, 2008, 3, e1628). An inhibitor of BMP1, TLL1 and/or TLL2 could therefore be beneficial in diseases where muscle function or muscle mass is diminished, including muscular dystrophy, sarcopenia, and cachexia associated with, e.g., heart failure, CKD, COPD, cancer or old age.
Taken together, the biology of BMP1, TLL1 and TLL2 lends strong support for their key roles in collagen processing, assembly and cross-linking, leading to the formation of a fibrillar collagen network that maintains tissue integrity and proper cellular microenvironment. This family of proteins may also play important roles in the etiology of fibrotic conditions, for example in the heart, lung, skeletal muscle, kidney, liver, skin, vasculature, nervous system, and eye, and inhibitors of these metalloproteases may provide broad benefits as anti-fibrotic agents for the treatment of diseases associated with fibrosis, such as myocardial infarction, heart failure, cardiac arrhythmias, hypertrophic cardiomyopathy, chronic kidney disease (CKD), post-acute kidney injury, diabetic nephropathy, delayed graft function post-transplantation, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), liver cirrhosis, non-alcoholic steatohepatitis (NASH), muscular dystrophies (e.g., Duchenne, Becker, limb-girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreifuss), glaucoma, corneal scarring, keloids, wound healing, adhesions, hypertrophic scarring, other scarring, e.g. associated with burns, surgery or other trauma, stroke, collagen vascular diseases such as systemic lupus erythematosus, rheumatoid arthritis and scleroderma, spinal cord injury and multiple sclerosis. Furthermore, BMP1, TLL1 and TLL2 inhibitors may have additional therapeutic applications in muscular disease based on their impact on myostatin biology, in particular muscular dystrophies (e.g., Duchenne, Becker, limb-girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreifuss), sarcopenia, and cachexia associated with, e.g., heart failure, CKD, COPD, cancer or old age.